Octet qk biosensor

SUMO1 (Boston Biochem UL-712) was biotinylated using NHS-LC-LC-biotin (succini-midyl-6-[bio-tinamido]-6-hexanamidohexanoate) (Thermo Scientific) at a 5:1 molar ratio of biotin to protein for 30 min at 25°C followed by rapid exchange into HBS-T (10 mM HEPES, pH 7.4, 150 mM NaCl, 0.05% Tween 20) by passage over a rapid desalting column. Conditions were chosen according to the manufacturer so that each protein was likely randomly biotinylated at an average of 1–2 positions. All BLI measurements were made on a FortéBio (Menlo Park, CA) Octet QK biosensor using streptavidin sensors. Assays were performed in 96-well microplates at 25°C. All volumes were 200 μL. After loading biotinylated ligand onto SA sensors, a baseline was established in buffer alone prior to association at varying analyte concentrations for 300 s. After the association phase, sensors were moved to buffer only to monitor dissociation for another 300 s. Nonspecific binding to sensors without ligand was negligible. Reference subtracted raw data were fit with a single-state global association-then-dissociation model using GraphPad Prism 7.01.

All biolayer interferometry measurements were carried out on a FortéBio (Menlo Park, CA) Octet QK biosensor with streptavidin sensors at 25°C on 96-well opaque plates. All volumes were 200 μl. Biotinylated CPP constructs were loaded for 300s after which sensors were moved to binding buffer only and a baseline was established. Association and dissociation phases were 300s each. Raw data were reference subtracted against the signal from a ligand-loaded sensor against buffer only and were then fit using a global one-state association-then-dissociation model with GraphPad Prism 5.03, from which kinetic and affinity constants were determined. Nonspecific binding was measured with respect to the response of 1 μM analyte protein against a sensor without ligand and was found to be negligible in all cases. Observation of rapid dissociation in the absence of calcium was accomplished by movement of the sensors into wells containing binding buffer with 10 mM EDTA and measuring nm shift for 300 s.